(1) Field of the Invention
Some types of viruses carry on their surface a material called HA antigen (Hemagglutination antigen) which agglutinates animal erythrocytes. In hemagglutinatin of HA antigen it is well known that certain kinds of viruses agglutinate erythrocytes of the same corresponding kinds of animals. For instance, a rubella virus agglutinates a one-day-old chick's erythrocytes as well as goose erythrocytes, an influenza virus agglutinates chicken erythrocytes (one-day-old and adult), a parainfluenza virus agglutinates guinea pig and chicken erythrocytes and a measles virus agglutinates monkey's erthrocytes. Accordingly, viruses can be identified and quantified by utilizing the above property.
The present invention relates to a preservative solution for fixed erythrocytes employed in a hemagglutination reaction (HA reaction) and a hemagglutination inhibition reaction (HI reaction) based upon the above hemagglutination of viruses. The HA and HI reactions have generally been utilized in diagnosing viral disease in the field of clinical testing.
(2) Description of the Prior Art
The HA reaction is employed in identifying a virus and determining its titer by utilizing the above specific antigenicity of several kinds of viruses. The HI reaction is utilized in determining an antibody against the HA antigen, that is which, HI antibody an organism acquires as a result of an immune response against a viral infection. Both reactions need erythrocytes as a reaction reagent. Though the use of fresh erythrocytes is preferable in order to make the agglutination or the agglutination inhibition reaction clear and accurate, this is practically impossible since it is not easy to get fresh erythrocytes on each test. Therefore, such a preservation method has been studied as the potency of erythrocyte for agglutination is not diminished even after longterm storage. For example, methods such as fixation with formalin etc., freeze-preserving or freeze-drying of fixed erythrocytes and so on are exemplified. In freeze-drying, further stabilization of erythrocyte has been attempted by such a treatment before the freeze-drying as washing which removes salt from the erythrocyte (JPN. Pat. NO. 50-37724), addition of glucose or serum albumin (JPN. Unexamd. Pat. Publn. No. 54-23119), and so on. However, a longterm preserving method for fixed erythrocytes in solution without freeze-drying has not yet been developed.
In developing a preservative solution able to keep erythrocytes stable for a long time without decreasing their ability to be aggultinated first we intended to elucidate the cause of nonspecific inhibition of the agglutination which restricts specific hemagglutination of rubella virus and consequently makes its result inaccurate. As a result, it was elucidated that the nonspecific inhibition of agglutination is caused by a phosphate buffer solution or a physiological salt solution which is usually used as a preservative solution for erythrocyte. Namely, the use of these solutions causes the exudation of .beta.-lipoprotein from the fixed erythrocytes to nonspecifically inhibit hemagglutination. Therefore, if the fixed erythrocytes contain .beta.-lipoprotein, in the rubella HI antibody titer test a negative specimen (antibody titer less than 8 times ) may be misjudged as positive and accordingly the judgement by the test becomes impossible.
In general, erythrocytes fixed with aldehydes can be preserved for a long time. They are, however, destroyed by a mechanical stimulation such as shaking, mixing etc. and then .beta.-lipoprotein exudes into an erythrocyte suspension. The reason why a lot of .beta.-lipoprotein exudes is deduced that, where fixed erythrocytes are preserved in such a usual aqueous solution containing various salts such as physiological saline and the like, erythrocytes are strongly adhered to each other under the influence of the salts and consquently they must be placed in a mechanically more stimulated condition for a long time in order to efficiently disperse them before use.